Lab 7

Lab 7: Pre-Lab

Your task in Lab 7 is to carryout your experiment regarding the rate of cellular respiration and begin to analyze your data. To prepare for Lab 7, please review this pre-lab page. Once you feel confident regarding the below topics, complete the corresponding LABridge in Blackboard.

Introduction/Review
Do you know enough?
What will we do in lab?
LABridge

Cellular Respiration

CONNECTION ALERT! Cellular respiration is the topic of Chapter 9 in your BIOL 120 lecture. Please review your textbook as needed for this lab.

Energy is the currency of life: all living organisms require energy to survive and reproduce. Metabolism is the series of reactions and processes, catalyzed by enzymes, which together maintain life. These reactions fall into two types: catabolic or anabolic. These processes are the inverse of each other and in photosynthetic organisms occur in tandem as the anabolic reactions of photosynthesis create the products that are then broken down by the catabolic reactions of cellular respiration (view figure at left).

There are two general classes of cellular respiration that are characterized by their relative efficiency (ATP production): anaerobic (without oxygen) and aerobic (oxygenated) respiration. We are focusing on aerobic respiration in this lab, which is a highly efficient process occurring within the mitochondria of eukaryotic organisms that have higher energy requirements for survival. In a 4 step process, oxygen and glucose are used to produce energy (ATP), H2O, and CO2,

Glycolysis ("splitting of sugar"): This step happens in the cytoplasm. One Glucose (C6H12O6) is broken down to 2 molecules of pyruvate. Requires 2 ATP to start, produces 4 with a 2 ATP net payoff.
Pyruvate Grooming: The pyruvate from glycolysis is shuttled into the mitochondria, where it is converted to a molecule called Acetyl CoA for further breakdown.
The Citric Acid Cycle: Occurs in the mitochondrial matrix. In the presence of oxygen (O2), all the hydrogens (H2) are stripped off the Acetyl CoA, two by two, to extract the electrons for making ATP, until there are no hydrogens left - and all that is left of the sugar is CO2 - a waste product - and H2O. The Citric Acid Cycle results in the production of only ~4 ATPs, but produces a lot of NADH, which will go on to the next step.
The Electron Transport Chain and Chemiosmosis ("the big ATP payoff"). Occurs in the christae of the mirochondria, the folded membranes inside. Electrons from hydrogen are carried by NADH and passed down an electron transport chain. The energy release runs pumps that creat an electrochemical gradient of H+ ions. In Chemiosmosis, a big pump called ATP Synthase uses the gradient to produce A LOT of ATP. Results in the production of ~28-32 ATPs for every molecule of glucose.

Review the steps of anaerobic and aerobic respiration by clicking the links in the sidebar.

Be sure you know and understand the generalized equation for aerobic cellular respiration.

ANAEROBIC Respiration review

Aerobic respiration review

Do you know enough about crayfish?

-From the Kentucky Wildlife Action Plan (WAP)

Abundance. There are 640 species of freshwater crayfish (Order Decapoda) worldwide (Crandall and Buhay 2008). More than 360 species are in the US and the southeast is known as a hotspot of crayfish diversity (Taylor et al. 2007). Kentucky is inhabited by 54 species, with some under taxonomic review and others potentially awaiting discovery. Seven species are endemic to the state of Kentucky.

Status. Freshwater crayfish are valuable indicator species of watershed health. They act as "canaries in the coal mine" and provide early warnings of ecosystem distress when populations decline. Modification of habitats, sedimentation, and dams are serious threats to freshwater crayfishes. Nationally, about 48% of crayfish species are of conservation concern (ranging from Vulnerable to Endangered); over a third (37%) of the Kentucky fauna falls into this category (KSNPC, 2010). Cave species are particularly at-risk from upland activities that pollute groundwater flowing into cave systems; this includes issues with chemical spills, agricultural runoff, salt from roads, and siltation from poor land use.

Review the links in the sidebar: Facts on KY Crayfish and Crayfish Natural History. Also, watch the short video below to see these guys in action!

Facts on KY Crayfish

Crayfish Natural History

What will we do in lab?

Your task was to design a proposal in Lab 6. In lab 7, we will conduct an experiment that tests the affects of one variable on the rate of cellular respiration in crayfish (AKA craw-fish or crawl-dad). Like most animals, they rely on aerobic cellular respiration to meet their energy demands.

Review the general procedural steps we will use below. Your exact methods will differ depending on which variable you have selected to test. Make sure you are comfortable with concepts below.

How will we measure the rate of cellular respiration? ...By measuring the CO2 produced as a bi-product of the metabolic process! You will place your crayfish in an aquatic environment and allow it to "respire." After a set period of time (decided by you). Then you will remove the crayfish and analyzed how much CO2 was produced as a result of cellular respiration.
Review the following steps to understand how we will quantify the respiration rate.
The water you will place your crayfish will be slightly basic and will appear pink! This is because it will contain a pH indicator called, phenolphthalein, which is pink in the presence of a base and turns clear when it approaches neutral (pH=7).
As cellular respiration is occurring inside your crayfish , inside the beaker, CO2 will be expelled into the solution as a bi-product. In an aqueous environment, this CO2 breaks down into carbonic acid (H2CO3) then into bicarbonate (HCO3-) and H+ ions.
Increased concentrations of H+ ions increase the acidity in solution and lower the pH. As the pH decreases the solution will turn from pink (basic) to clear (neutral).
BUT! We will not wait for that to happen on its own, it would take far too long!
So...after your crayfish "respires" in solution for your set period time, you will remove it. The water will still appear pink. Some respiration and CO2 production HAS occurred, but not enough to change the pH to see a color change. So we would add more acid to the solution (H2SO4), via titration, until we see a color change from pick to clear. View Diagram.
You will use the "amount of titrant used" to calculate the micromoles of carbonic acid (H2CO3) produced in solution using this equation.
Lastly you will complete your analysis using group or class data including creating a bar graph and using an unpaired t-test calculator to test your hypothesis.

Review the YouTube video below on titration with phenollphthalein so you have a good idea of what to expect in lab!

How pH change indicates respiration rate. Click to enlarge.

Special Note! You have no doubt noticed the importance of sample size for this experiment to work well. Depending on your lab size, timing, and proposals, you may be asked to share data as a class or among several groups testing the same variables. You may also be asked to pick just one variable and all test it together as a class.

Be sure you understand the relationship between the amount of titrant required and the rate of cellular respiration.

Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!

Lab 7: Protocol

In today's lab you will work with your lab group to conduct your experiment and begin to analyze your data.

Exercise I. Review your research proposal and the pre-lab and revise where necessary

Exercise II. Conduct your experiment & collect data

Exercise III. Analyze your data

Lab Objectives: Following today's lab, you should be able to...

Exercise I
Exercise II
Exercise III

Exercise I. Prepare

Procedure.

Open the Lab 7 Notebook Guide
Open your research proposal on Cellular Respiration from Lab 6.
Review the pre-lab.
At this point your class might decide to work together to narrow down your choices. If you work together as a class in large groups, you can increase your sample size.
Review the slide show (in Lab 6) on your experimental protocol as needed. for a refresher.
Complete Exercise I in the the Lab Notebook Guide. Ask the following questions and augment/revise your proposal as necessary.
1. Hypotheses remain the same?
2. Independent, dependent and confounding variables remain the same?
3. How long does your crayfish need to respire? If you decide to test temperature, please note that the ice will need to sit in your beaker for at least 5 minutes, before adding your crayfish so the temperature can stabilize.
4. How many trials will you do?
5. Do you need a control? (YES) It will either be testing a crayfish in solution without glucose (vs. with) -OR- at room temperature (vs. cold).
6. What kind of statistics will you use?
7. What kind of graph might you make?
Safety Note: You will need to wear your gloves and goggles all day. Sulfuric acid is dilute but requires caution. All shellfish allergies should be reported.
Animal Treatment: Any cruel treatment of animals in BIOL 121 will not be tolerated. Be careful. Be respectful. Do NOT titrate your crayfish. Do NOT put your crayfish on its back.
Before moving on, decide who will be responsible for what. Refer to your contract. You'll need a careful and responsible timer and a slow and steady titration team.
Make sure some is taking pictures for use on your poster!
STOP. You must get your instructor's "OK" before you can move-on. She/he will need to go over your Lab Notebook Guide before you proceed.

Remember: Basic solutions are pink with the phenolphthalein indicator and acidic solutions are clear. The crayfish will expel CO2, making the solution more acidic and closer to "clear."

Lab 7 Notebook Guide. Click to download.

Takes pics for your poster!

Exercise II. Run your experiment & collect data

With an understanding of cellular respiration and how the phenolphthalein indicator works, we should be able to determine the rate of respiration of our crayfish in different conditions. You will complete 3 titrations/tests today in lab:

Baseline: This test will establish how much titrant is required to reach our endpoint (from pink to clear) without a crayfish. The value you are aiming for will be written on the board (Nb)
Experimental Control: Conduct your experimental control (condition 1) with a crayfish in a phenolphthalein solution WITHOUT Glucose/ ROOM Temperature (Ne or Ne1).
Experimental Treatment: Conduct your experimental treatment (condition 2) with a crayfish in a phenolphthalein solution WITH glucose, or in a COLD solution (Ne or Ne2).

Materials.

Gloves & goggles all day

Crayfish

Phenolphthalein Indicator

Bench Set-Up

Graduated Cylinder

Glassware

Burette & Titration Stand

Ice Bath

Stock Solution (Pheno)

Pitchers for Solution

Titration Flasks

Test Beakers

Procedure.

For the purposes of time, you will start with your experimental control (condition 1), then practice your titrations and establish a baseline, then do your experimental treatment (condition 2). Lastly, you will do some calculations. This protocol has been separated into four parts (A-D). Be sure to follow the directions closely as we move from one section to another to take advantage of your wait times. *Items in pink below can vary based on the size of our crayfish each term and will be provided by the TA.

Part A) Practice Titration and Establish your Baseline without a Crayfish (Nb)

Procedure. You will practice titration and establish a baseline (without crayfish).

Identify your burette, stand, titrant, flasks and beakers.
Practice using the burette. Place either flask underneath and play with the tap. Vertical = completely open. Horizontal = completely closed. Find the position that allows for a steady drip of the titrant.
Once you feel confident, ask your TA to place your used titrant back into the burette.
Before you titrate for the baseline, place a white index card under the flask to help you see the color change better.
You are working to slowly remove the pink color. As the color lightens, you should start to slow. Only when the pink is gone, and stays gone, have you reached the endpoint (see image below).
Place 50mLs of the stock solution (from your pitcher) into a flask (either one is fine).
Record the starting volume of your burette.
Titrate slowly and steadily until you reach a clear endpoint. Your values should come close to those on the board.
Complete your acid-base titration to clear endpoint.
Record the ending volume of your burette.
Calculate the amount of titrant you used to reach the clear endpoint. This is your baseline (Nb). You will use it to calculate the umols of CO2 released by your crayfish in your two test conditions.
Go into your data collection excel sheet and label your two conditions properly, in the first cell of each column.
Record this value as the baseline (Nb) for each condition in your data table.

Lab 7 data collection excel sheet. Click to download.

Click to enlarge.

What to look for when approaching your endpoint.

Part B) Experimental Control (condition 1) "WITHOUT Glucose" or "ROOM Temperature" with Crayfish (Ne1)

Procedure. Set-up and begin condition 1, your experimental control.

Obtain a crayfish.
Size matters here. Some terms our crayfish are larger than others. Follow your TA's instructions on the volume required.
Place 100mL of stock solution into a graduated cylinder. Record the volume.
Hold the gradated cylinder at an angle and gently slide the crayfish in tail first. You may need multiple people working together.
You may also need to gently guide the crayfish down to the bottom using your glass rod.
Gently tilt the graduated cylinder back upright.
Record the change in volume. Open your data collection excel sheet to tab one and record the volume of this crayfish under condition 1.
Transfer the solution and crayfish (gently) into your test beaker.
Your TA will demo this. You may need to add more solution to the beaker to ensure the crayfish stays submerged. This depends on the size of your crayfish and the level will be provided by your TA.
Set your time and begin the test. Your crayfish will be allowed to respire into this beaker for 20m (0.33h) or 30m (0.5h). Check your TA.
Make sure someone is timing.

How to determine crayfish volume. Click to enlarge.

Part C) Experimental Treatment (condition 2) "WITH Glucose" OR "COLD Temp" (Ne2)

Procedure. Set-up and begin condition 2, your experimental treatment condition.

Obtain the appropriate treatment solution depending on your test.

If you are testing for glucose, obtain the stock solution with added glucose. It will be labeled on the large white bucket. Put 50-100mLs (as needed to submerge your crayfish) in beaker 2.
If you are testing temperature, obtain the stock solution. Put 50-100mLs (as needed to submerge your crayfish) in beaker 2. Put the beaker on an ice bath (fill a second container with ice and sit your beaker on top). You must wait 5-10 minutes for this solution to get cold before adding your crayfish. See the photo in the sidebar.

Obtain your crayfish. Determine and record its' volume using the same protocol as in part A (#4-9). Transfer the solution and crayfish (gently) into your test beaker as you did before.
You must keep your crayfish beaker ON ICE for the entire test.
You may need to add more solution to the beaker to ensure the crayfish stays submerged. This depends on the size of your crayfish and the level will be provided by your TA.
Set your time and begin the test. Your crayfish will be allowed to respire into this beaker for 20m (0.33h) or 30m (0.5h). Check your TA.
Be sure someone is recording this time as well.

How to determine crayfish volume. Click to enlarge.

Ice Bath Set-Up if testing temperature.

Part D) Determine Titrant Required and Calculate the Carbonic Acid (umols) Produced by your Crayfish

Procedure I. Determine the titrant Required for Each Condition

Click to enlarge.

What to look for.

Check the time remaining on each test.
Once test 1 is complete, remove your crayfish from the test beaker and place it (gently) in the recovery tank on the front desk.
You will use 50 mL of your control or experimental solution for the titration. Carefully transfer 50mLs into your condition 1 flask.
Record the starting volume of your burette.
Complete your acid-base titration to clear endpoint.
Record the ending volume of your burette.
Calculate the amount of titrant required for condition 1. Record it as the Ne for condition 1 and complete the data table.
Once test 2 is complete, remove your crayfish from the test beaker and place it (gently) in the recovery tank on the front desk.
You will use 50 mL of your control or experimental solution for the titration. Carefully transfer 50mLs into your condition 1 flask.
Record the starting volume of your burette.
Complete your acid-base titration to clear endpoint.
Record the ending volume of your burette.
Calculate the amount of titrant required for condition 1. Record it as the Ne for condition 1 and complete the data table.

Procedure II. Determine the Carbonic Acid (umols) Required for Each Condition

Be certain you are using the correct variables and correct units.

Clean your station before checking out.

Use the equation in the sidebar to calculate to umols of carbonic acid produced by your crayfish in each condition/test.
Be certain you are using the correct variables. V and Ne will be different for each condition's respective calculation. T is in hours, for example 30m = 0.5.
Once you have completed the calculations, check them against the auto-generated calculations on tab 1 of your data sheet.
Add your data to the class or group data on the board or Google doc.
Open the second tab in your Excel spreadsheet labeled "Class or Group data."
Enter in the class or group data from the board into each column. Remember these values are in umol of carbonic acid produced by the crayfish in two groups (i.e., in the two different treatment conditions).
Be sure to clean your stain before you check it and wipe it down.
Leftover titrant stays in the burette and
All other solutions can go down the drain.

REMEMBER why we have to use titration! In an aqueous environment, CO2 combines with water to first create carbonic acid, which is then broken down into hydrogen ions. The addition of H+ ions results in a change in pH: the pH decreases and becomes more acidic. The introduction of CO2 from the crayfish would have lowered the pH some, but not enough in 20 minutes to make it acidic enough to turn clear. The CO2 released is transformed to carbonic acid which we can measure with this equation.

Exercise III. Analysis

Comparing the single data points you have is an OK way to test for differences in respiration rate. But, ideally we would have a larger sample size (using class data) and statistics. Now, what type of statistics should we use to determine if the mean respiration rate between our 2 groups is significantly different? ...hopefully this is an easy answer. A T-test! We need to determine if the mean umols carbonic acid (H2CO3) produced in your two conditions are close enough to be the same or far enough a part to be different.

Remember: Statistics solve the problem of determining if "more" or "higher" or "different" than is actually enough to be important and biological relevant. Using the principles of probability, they help us parse what we observe from randomness (chance alone) as meaning (a real difference, or a real relationship). Statistics tell us how likely we would be to make the same observations we have made, if chance and randomness were the only drivers. If the probability is very low (<5%), we refer to these patterns as significant.

Procedure.
Comparing the single data points you have is an OK way to test for differences in respiration rate. But, ideally we would have a larger sample size (using class data) and statistics. Now, what type of statistics should we use to determine if the mean respiration rate between our 2 groups is significantly different? ...hopefully this is an easy answer. A T-test! We need to determine if the mean umols carbonic acid (H2CO3) produced in your two conditions are close enough to be the same or far enough a part to be different.

Watch the t-test calculator demo in the sidebar.
This video goes over using the t-test calculator for both a paired and unparired test. Start the video around minute 7.10 for a demo on an unpaired t-test.
Pay close attention for how to use a one-tailed version, which is the most appropriate for this experiment and your hypothesis.
After you complete your unpaired t-test, create a matching bar graph.
Watch the demo in the side bar.
Pay close attention the titles of your axes and how to create your error bars.
Complete your Lab Notebook Guide.

Visit the stats section of our research library.

Bar Graph Demo

t-test CALCULATOR demo

T-test Online Calculator.

To pair or not to pair?
If the data are extremely inconsistent, try a paired T-test to account for differences between group calculations.

Lab 7 BIOL 120 CONNECTIONS
Section 1.6: Doing Biology
Big Picture 1: How to Think Like a Scientist
BioSkillls 2: Reading & Making Graphs
BioSkillls 3: Interpreting Standard Error and Using Statistical Tests
BioSkillls 4: Working with Probabilities
Chapter 9: Cellular Respiration

Faculty Spotlight: Dr. Noah Ashley

[email protected]

We are conducting experiments to identify factors that affect the physiological process of cellular respiration in crayfish. Similarly, Dr. Noah Ashley's lab works to identify physiological, immunological, and behavioral responses to various factors, like sickness and sleep loss, in mice and birds. Specifically, they are investigating the costs and benefits of the sickness response in vertebrates, the inflammatory response in sleep-deprived mice, sleep loss in migratory birds, and the sleep-wake cycle in arctic songbirds. Dr. Ashley's lab is extremely productive! His research proposals have been funded by the NSF and the NIH. Learn more here (Lab Web Page). You just might recognize one of his current graduate students!

Research Key Words: physiology, ornithology, eco-immunology, genomics, sleep loss, arctic song birds, migrating birds
Recent Publication: Cooper, L. N., Mishra, I., Ashley, N.T. 2019. Short-Term Sleep Loss Alters Cytokine Gene Expression in Brain and Peripheral Tissues and Increases Plasma Corticosterone of Zebra Finch (Taeniopygia guttata). Physiological and Biochemical Zoology 92:80-91.

​[Metabolism]Lab 7: What factors affect the rate of cellular respiration in crayfish?